1. Field of the Invention
The present invention relates generally to neurobiology. More specifically, the present invention relates to molecular engineering of monoamine oxidases to determine those domains that play a role in metabolizing cellular neurotransmitters and vasoamines.
2. Description of the Related Art
Monoamine oxidase A and B (MAO A and B, EC 1.4.3.4) are integral covalent flavoproteins of the outer mitochondrial membrane in almost all tissues of mammals (1). MAO A and B catalyze the oxidative deamination of biogenic and vasoactive amines and the oxidation of xenobiotics (2, 3). These isozymes play important roles in regulating the intracellular level of various amines in the central nervous system and peripheral tissues of mammals and in protecting those cells and neurons that non-specifically take up neurotransmitters where they have no legitimate physiological function (4). Several psychiatric and neurological disorders (i.e., Parkinson""s disease, depression, and alcoholism) have been linked to these enzymes (5-7).
MAO A and B are distinct polypeptides, but they have a high degree of identity in their nucleotide and deduced amino acid sequences (8). They also differ in substrate and inhibitor preferences and in cell and tissue distributions (4, 10-13). One FAD molecule is covalently linked at Cys397 in each subunit (58.8 kDa) of human MAO A and B (9). Previous studies demonstrated that there are two FAD binding sites near the N-terminus (residues 6-34 and 41-45) that are required for both covalent and noncovalent binding of FAD and the generation of MAO B catalytic activity (14-18). In contrast, variants with substituted residues near the middle of the sequence (residues 222-227) can bind FAD noncovalently but not covalently. These variants also have reduced MAO B activities (18).
Based on these observations, it was proposed that FAD is coupled to the MAO B polypeptide in a stepwise process, beginning with the FAD-binding sites near the N-terminus where an initial topological dock for FAD is provided (18). FAD is then delivered to the FAD binding sites in the middle of the sequence to provide another topological dock that further secures FAD. Finally, FAD is delivered to Cys397 in a position which places the 8xcex1-methyl group of the isoalloxazine ring of FAD in close proximity to the thiol group of Cys397 to facilitate covalent flavinylation.
The prior art is deficient in identifying the active site of the monoamine oxidase (MAO) B enzyme and the effect that mutations to this site have on enzyme activity and the regulation of neuro- and vaso-amines. The present invention fulfills this long-standing need and desire in the art.
Monoamine oxidase A and B (MAO A and B) are major neurotransmitter- and xenobiotic-metabolizing enzymes that are thought to play a role in psychiatric and neurological disorders. These isozymes have a high degree of identity in their deduced amino acid sequences, but are encoded by different genes on the X-chromosome. Previous studies on MAO B have shown that FAD binds both noncovalently and covalently to specific amino acid residues that reside in highly conserved regions of MAO A and B.
Comparison of the deduced amino acid sequences of MAO A and B from different species (8, 22-25) reveals three highly conserved regions. These regions reside near the N-terminus, in the middle of the polypeptide, and on each side of the covalent FAD binding residue (Cys397) in MAO B. It is hypothesized that the highly conserved region flanking Cys397 also plays a role in the covalent binding of FAD. This notion is supported by Wouters and Baudoux (20), who constructed the first partial three-dimensional model of human MAO A based on a combination of primary sequence analysis, secondary structure determination, fold recognition studies, and knowledge-based modeling. This model predicted that Tyr407 (corresponding to Tyr398 in MAO B) is the only amino acid residue that resides in close proximity to the isoalloxazine ring of FAD in the highly conserved region flanking the covalent FAD binding residue (Cys406) in MAO A. Furthermore, Mauch et al. (21) found that Arg67 in the sequence Arg67-Ser6-Gly69-Gly70 (SEQ ID No.2), which immediately precedes the covalent FAD binding residue (His71), is obligatory for covalent flavinylation of 6-hydroxy-D-nicotine oxidase (6-HDNO). This sequence pattern has also been found in several other flavoproteins with a covalent histidyl(N3)-8xcex1-FAD linkage (FIG. 1B) (21). It is proposed herein that the sequence Tyr393-Ser394-Gly395-Gly396 (SEQ ID No. 3) immediately preceding the covalent FAD binding residue (Cys397) plays a similar role in MAO B as does Arg67-Ser68-Gly69-Gly70 (SEQ ID No.: 2) in 6-HDNO (FIG. 1). To test this hypothesis, variants of human MAO B were made at Tyr393, Ser394 and Tyr398 to determine if these residues participate in the covalent flavinylation of human MAO B. Analysis of these variants demonstrated that the aromatic moieties at Tyr393 and Tyr398 are essential for covalent FAD binding and MAO B catalytic activity.
In this study, it is demonstrated that the aromatic moieties at Tyr393 and Tyr398, which are located on each side of the covalent FAD binding residue (Cys397) participate in covalent FAD binding, and the generation of catalytically active MAO B. Based on these results and a three-dimensional model of MAO A (20), it is proposed that two antiparallel xcex2-strands flank the covalent FAD binding residue (a cysteine) in flavoproteins with a covalent cysteinyl(S)-8xcex1-FAD linkage and that this structural motif plays an important role in covalent FAD binding in MAO B.
One object of the present invention is to provide isolated, genetically-engineered MAO B enzymes having at least one amino acid substitution for amino acids near the wild type MAO B flavinylation site, where the wild type amino acid is Tyrosine at position 393, Tyrosine at position 398 and Serine at position 394. The present invention additionally provides isolated DNAs that encode these genetically-engineered MAO B enzymes, and plasmids containing these DNAs along with regulatory elements necessary for expression of these DNAs in a cell.
Specific embodiments of this object of the present invention include the following substitutions: where if the wild type amino acid is Tyrosine 393, the amino acid substitution is Alanine or Phenylalanine; where if the wild type amino acid is Tyrosine 398, the amino acid substitution is Alanine or Phenylalanine; and, where the wild type amino acid is Serine 394, the amino acid substitution is Alanine.
An additional object of the present invention is to provide pharmaceutical compositions which interact with the active site of MAO B. Specific compositions include derivatives of active site components, such as FAD, 2xe2x80x2-deoxy FAD and 3xe2x80x2-deoxy FAD, and derivatives of mechanism-based inhibitors that belong to the acetylenic and cyclopropyl amine classes.
Additionally, an object of the present invention is to provide a method for regulating MAO B comprising the step of mutating an amino acid in the MAO B active site. Specifically, the amino acid to be mutated is selected from the wild type amino acids Tyr 393, Tyr 398, or Ser 394.
Another object of the present invention is to provide a description of the active site of monoamine oxidase B, such that pharmaceutical compositions can be designed to interact with the active site. Specific embodiments of this object of the invention include 2xe2x80x2-deoxy FAD, 3xe2x80x2-deoxy FAD, and derivatives of deprenyl (phenylisopropyl-methylproinylamine) and trans-phenylcyclopropylamine. Molecular modeling is applied to determine which derivatives are most likely to interact with components in the active site of the enzyme.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments. These embodiments are given for the purpose of disclosure.